Bacterial vaccines with p-hydroxybenzoates and their production



3,035,980 Patented May 22, 1962 3,035,980 BACTERIAL VACCINES WITH p-HYDROXYBEN- ZOATES AND THEIR PRODUCTION Howard Tint, Havertown, and John H. Brown, Marietta,

Pa., assignors to American HomeProducts Corporation, New York, N.Y., a corporation of Delaware N Drawing. Filed Oct. 5, 1960, Ser. No. 60,556

10 Claims. (Cl. 16778) This invention relates to vaccine compositions and more particularly to detoxified compositions containing one or more vaccines, and their method of preparation.

Certain vaccines prepared from killed organisms contain toxins which must be removed or neutralized prior to use. Actually, it has been found that vaccines made from killed and suspended organisms whihc had been previously detoxified in the course of their preparation develop toxins on shelf storage. Apparently, even though the organisms are killed, there still remains some mechanism which will produce or liberate toxins. Noteworthy in this respect are the organisms Hemophilus pertussis and Salmonella typhosa.

It is well recognized that toxic matter must be removed or inactivated from vaccines inherently contained or developed in them since such vaccines will not only produce local reactions, such as swelling and inflammation, at the site of application but will frequently produce a general malaise and fever lasting for a considerable period of time. Indeed the necessity for the removal or inactivation of toxic elements is so well recognized that special tests approved by the National Institutes of Health have been designed to indicate if a product is too toxic or dangerous for safety. However, after a product has passed the necessary safety tests for toxicity, there is no good way to prevent the development of additional toxic elements on storage. The need exists, therefore, for a simple and relatively inexpensive way to prevent the formation of, as well as to remove or neutralize, toxic elements in a vaccine.

Prior art suggestions or procedures for detoxification have not proved completely satisfactory. In an evaluation of the known procedures for detoxification of pertussis vaccine, perhaps the most troublesome of vaccines in this respect, one author, reporting in the Journal of Immunology, volume 69, pages 201-216 (1952), advocated heating the vaccine at 56 C. for 30 minutes or 1 day at 34 C. over any other procedure such as the use of special chemical agents, i.e., thirnerosal, phenol and formalin or a physical agent, namely, ultraviolet irradiation, all of which were found to cause substantially lowered potency or antigenicity. Actually, even though this investigator selected heating as the best method for detoxifying pertussis vaccine, it has been found that detoxification by heat alone is unreliable and reproducibility is uncertain. Moreover it has been found that heating often produces a considerable loss in potency when operating in relatively large batches.

In order to overcome the deficiencies of detoxification as previously advocated, it is common practice to first detoxify a vaccine of H. pertussis, for example, or even one of the multiple vaccines, by either heat or irradiation or by one of the chemical methods previously suggested, and then to age the vaccine for a considerable period of time, generally three months or longer, under refrigeration conditions (5 C.). Such a procedure results in a large and uncertain inventory position since one is never sure when a bacterial suspension will have detoxified to a level satisfactory from a health standpoint. One batch may detoxify in a few weeks and another will require storage for two years before it detoxifies to an acceptable health level.

The present invention is based on the discovery that the alkyl esters of p-hydroxybenzoic acid or the alkali salts thereof, hereafter broadly referred to as parabens, are not only antibacterial which is well known but, in certain concentrations, are highly effective detoxification agents for vaccines produced or prepared as suspensions of killed pathogenic organisms, i.e. H. pertussis, S. typhosa, S. schotmailleri, S. paratyphi, E. coli, Staph. aureus, Staph. albus, M. catarrhalis, etc. In general, it is known that suspensions of killed pathogenic organisms develop toxins on storage and there is a clear need for a means to inactivate such toxins within a reasonable time. The parabens suitable for both killing and detoxifying are the lower alkyl benzoates, preferably the methyl, ethyl and/ or propyl-p-hydroxybenzoates. These compounds will be hereinafter specifically designated by the particular alkyl group in the ester radical as, for example, methylparaben. It has been found that in the presence of one or more of these parabens, the potency remains high and, more importantly, the parabens permit a much shorter period for detoxifying in the course of manufacture and their effects are reliable and reproducible with successive batches. Thus, where prior art proce-. dures resulted in a product that would take from days to years to detoxify, the use of the parabens results in products that can reliably detoxify in hours or a fraction of the time needed previously.

The invention is carried out by first preparaing a suspension of the organism or organisms to be killed and detoxified in the usual manner for preparing vaccines. Briefly, this involves growing the organism on an agar growth medium slant, for example, a Bordet-Gengou slant, at incubating temperature. When the growth is adequate, it is suspended in saline and used to inoculate a large amount of liquid growth medium and the organism is grown to the extent deemed necessary for obtaining a vaccine suspension. In the case of H. pertussis, for example, growth is carried out until a suspension of approximately 20-30 opacity units can be obtained.

When sufficient growth of the organism is obtained, the suspension is now ready for killing and detoxifying. This is carried out by adding to the suspension an alkyl ester or salt of p-hydroxyhenzoic acid or two or more of these together. The amount of detoxifying agent added may range from about 0.15 mg./cc. of suspension to the maximum amount that will solubilize in the suspension, which, in the case of propyl-p-hydroxybenzoate, is approximately 0.20 mg./cc. or, in the case of the methyl analog, is 2.5 mg./ cc.

It has been found that when a single paraben is used, the amount approaching the limit of solubility of the paraben in the vaccine gives the best results, namely, the shortest period of time necessary to achieve detoxification. However, when using a combination of parabens, it has also been found that even better results are achieved than that obtained in using a single paraben. With respect to amounts of parabens when used in combination, it is preferred to use each paraben to the limit of its solubility in the vaccine. Thus, with respect to the combination of methylparaben with propylparben, for example, the preferred amounts of each are 2.5 mg./ cc. and 0.2 mg./ cc. of vaccine respectively.

When the killing and detoxifying agent or agents have been added, the suspension is held for a period of time and tested at intervals until toxicity and viability have been reduced to safe limits. The desired action of the agent or agents added will take place satisfactorily at room temperature but, if carefully carried out to avoid loss of potency, the action may be somewhat expedited with heating, up to about 37 C. At room temperature, satisfactory detoxified batches may be obtained when the suspensions are held for as little as 16 hours. However, with extremely toxic batches, or where only one paraben ing the poliornyelitis vaccine.

3 is utilized at less' than maximum amount, killing and detoxification may take place in approximately two weeks. One may contrast this with similar batches containing no detoxifying agent which did not detoxify to safe limits even after 36 days. 7

As stated previously, a killed and detoxified vaccine using a paraben as described may be combined wth other vaccines. Thus, one may combine a paraben killed and detoxified H. pertussis vaccine with diphtheria toxoid and tetanus toxoid vaccine prepared in known manner, adding physiologic saline solution or adjuvants, if desired, to produce a triple vaccine containing in solution the alkyl ester p-hydroxybenzoates orsalts thereof. In the final product, the parabens act to maintain the sterility of the com position.

It is also advantageous to utilize the invention in the preparation of a multiple vaccine containing, as one of the ingredients, poliomyelitis vaccine concentrate. As an example, one may add the latter vaccine to either paraben killed and detoxified H. pertussis or more preferably to the triple vaccine mentioned previously. Again, the presence of the detoxifying paraben or combined parabens serves to preserve the combined vaccines against bacterial contamination without neutralizing or destroy- It is worthy of note that thimeros'al, normally used as a general preservative, has led to adeterioration in antigenicity of some poliomyelitis virus vaccines.

The following examples will serve to illustrate the invention in greater detail.

Example 1 An S. typhosa strain, identified as Boxhill 58, and paratyphoid strains A and B, identified as 41N-42 and 4lH-6, were separately incubated upon a nutrient agar substrate. The organisms were individually harvested and concentrated by suspending in saline, and to the suspension of each strain was added 2.5 mg/mL of methyland' 0.2 mg./ml. of propyl-p-hydroxybenzoates. All of the suspensions were held at room temperature (24 C.) for 144' hours.

Each of these strain concentrates was centrifuged at 3,000 rpm. at 5 C. for two hours and the heavier fractions were resuspended to original volumes with buffered saline solution.

A liter of vaccine was prepared by combining the above three components to make a product a containing 8.33 7

ml. of S. typhosn organisms, 1.82 ml. of paratyphoid A organisms, 2.84 ml. of paratyphoid B organisms, 10.00 mi. of the mixed benzoates in the statedratio, and 977 .01 ml. of buffered saline. The vaccine was held at refrigerature temperature and tests for viability and sterility at 96 hours from the time of benzoate addition showed complete killing and sterility. At 17 days from the time of benzoate addition, satisfactory detoxification was shown by acceptable animal test.

Example 2 Lyophilized H. pertussis seed was'recon'stituted in physiological saline. A Bordet-Gengou slant was planted with 0.2 ml. of the reconstituted seed and incubated at 34 C. When the growth appeared adequate, it was scraped into 2 ml. of physiological saline and used to inoculate 200 ml. of modified Cohen-Wheeler medium. After; about 24-hours at 34 C. the pH was adjusted to 6.5 and to approximately 18 to 20' opacity units.

An amount of methyi-p-hydroxybenzoate, equivalent to 0.5 rug/cc. of final product, diluted in sufiicient alcohol to result in 4 cc. per liter of vaccine, was added and the mixture allowed to stand at room temperature. Sarnples where withdrawn for viability and toxicity'tests, the latter performed according to N.I.H. requirements; The vaccine product showed zero viability and acceptable detoxification atabout 15 to 22d'ays;

4 Example 3' The same procedure as in Example 2 was carried out using 2.5 mg./cc. of methyl-p-hydroxybenzoate. The vaccine product showed zero viability and was detoxified in 24 to 40 hours.

Example 5 V The same procedure as in Example 2 was carried out using 1.5 mg./cc. of methyland 0.20 mg./cc. of propylp-hydroxybenzoates. The vaccine product containing the mixed benzoates showed zero viability and was satisfacton'ly detoxified in 16 to 24 hours.

Example 6 A fully detoxified H. pertussis vaccine as described in Example 5 was utilized to make a triple and quadruple vaccine containing the ingredients as listed below:

Approximate percentages of the final composition, percent by volume (1) Diphtheria toxoid concentrate, used undiluted 1.0 2) Tetanus toxoid concentrate; used undiluted 2.2 (3) Pertussis vaccine; harvest bulk concentrated apgrox. 2-fold to yield a level of. O.U./ 1 40.0 (4) olutions of aluminum chloride and sodium phosphate in saline. These levels, when mixed in the final preparation, yield approximately the desired final AIPQs content 40.0 (5) Physiologic saline 16.7

(approx) 100.0

The above formulation contains 1.7 mg/ml. of par-abens in the vaccine.

The following should be noted with respect to the above formula:

(1) Diphtheria toxoid is usually prepared from harvests at Lf levels ranging from 50 to 100 per ml. In refining this antigen by the metaphosphate procedure, the toxoid is eventually obtained as a concentrate of the order of magnitude of 3,000 Lf per ml.

(2) Tetanus toxoid produced at approximately 30 Li per ml. is prepared to a level of approximately 900 to 1,000 Lf per ml. in a concentrate.

. (3) The pertussis vaccine, produced at approximately 25'opacity units per ml., must also be concentrated (by centrifugation) to a level higher than the harvest level.

Lf is a designation of toxoid activity, and literally means flocculating unit. This unit signifies a certain antigenic capacity. The opacity unit for pertussis is a measure of the cellular content of the vaccine, and is also referrable to antigenic activity.

In preparing a quadruple vaccine, particularly one containing apoliovaccine ingredient, one merely replaces physiologic saline solution with the additional vaccine.

In the vaccines as given above there must be specific final contents in terms of the potency of the individual antigens, as well as a definitive amount of aluminum phosphate adjuvant, assuming the latter is desired. A typical formula is given below that is designed to contain the following ingredients on a mi. basis, with the understanding that each injection dose is 0.5 ml., and a total human immunizing dose represents a total of 1.5 ml., or a series of three injections:

' Triple antigen Antigens: with adjuvant Diphtheria toxoid Lf 30 Tetanus toxoid Lf 20 Pertussis vaccine O.U 20 Aluminum phosphate mg 2.6

(Alum equivalent) mg (10) We claim:

1. A paraben killed and detoxified vaccine of the toxin-forming bacterial type containing at least one paraben in a concentration of approximately the limit of its solubility in the vaccine but not exceeding about 2.5 mg./ml. of vaccine.

2. The composition of claim 1; wherein the vaccine comprises H. pertussis.

3. The composition of claim 1; wherein said paraben in the composition is methylparaben.

4. A paraben killed and detoxified vaccine composition comprising H. pertussis vaccine containing, for detoxification, about 1.5 mg./ml. of methylparaben and 0.2 mg/ml. of propylparaben.

5. A multiple vaccine comprising a paraben killed and detoxified H. pertussis vaccine component, diphtheria toxoid, tetanus toxoid, containing both methyl and propylparabens for detoxification, said parabens being present in a concentration approximately the limit of their solubility in said vaccine.

6. The composition of claim 5; wherein said vaccine includes poliovaccine.

7. The process of forming a suspended cell paraben killed and detoxified vaccine comprising incubating at toxin-forming bacteria in a suitable growth medium until an adequate concentration of said bacteria is obtained, and then killing and detoxifying said bacterial content by adding a lower alkyl paraben and storing the mixture until detoxification takes place, the amount of paraben used for detoxification being about the maximum amount capable of remaining in solution in the mixture.

8. The process of producing a para'ben killed and detoxified vaccine comprising incubating H. pertussis at about 34 C. in a suitable growth medium, adjusting the pH of the medium to approximately neutral and the antigenic activity to about 18 to 20 opacity units per ml., and then killing and detoxifying the bacterial content by adding a para'ben to said growth in an amount approximating the limit of its solubility in said medium and storing said composition until it has detoxified.

9. A vaccine composition as recited in claim 1; wherein the vaccine comprises S. typhosa.

10. A paraben killed and detoxified vaccine composition comprising S. typ-hosa and paratyphoid organisms combined with methyland propyl-p-hydroxy-benzoates for detoxification wherein, on reconstitution with an aqueous medium, the parabens are present in an amount of about 2.5 mg./ml. of the methylparaben and about 0.2 mg./m1. of the propylparaben.

References Cited in the file of this patent UNITED STATES PATENTS Behrens Dec. 15, 1959 OTHER REFERENCES 

1. A PARABEN KILLED AND DETOXIFIED VACCINE OF THE TOXIN-FORMING BACTERIAL TYPE CONTAINING AT LEAST ONE PARABEN IN A CONCENTRATION OF APPROXIMATELY THE LIMIT OF ITS SOLUBILITY IN THE VACCINE BUT NOT EXCEEDING ABOUT 2.5 MG/ML OF VACCINE. 